In bacteria, a widely used means of genetic regulation is a non-protein coding RNA element called a riboswitch. These RNA sequences are cis-acting elements found in the 5'-untranslated region (5'-UTR) of mRNAs and regulate gene expression via their ability to directly bind small molecule metabolites to a receptor domain. Binding of the ligand directs the folding of a mutually exclusive secondary structural switch in a downstream regulatory domain that directly interfaces with the expression machinery (either RNA polymerase or the ribosome). A variety of basic metabolic pathways including purine, amino acid, and cofactor biosynthesis in a number of bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis;in Bacillus and related Gram-positive species, over 4% of all genes are controlled in this fashion. Since these RNAs have already evolved to specifically bind small molecules, they have become of great interest as novel targets for designing therapeutics targeted against pathogenic bacteria. Towards the long-term goal of developing a molecular understanding of how riboswitches efficiently regulate gene expression, purine-binding riboswitches will be employed as a model system for addressing structural and mechanistic questions. This proposal details a set of specific aims that address: (1) how structural variation in the ligand receptor enables the regulatory response of individual riboswitches to be "tuned" to the gene it controls, (2) detail the structural plasticity of the RNA within the ligand binding site that allows it to be recognized by diverse purine analogs, (3) broaden our understanding into the mechanism by which ligand binding to the receptor is communicated to downstream regulatory domain to effect biological activity, and (4) determine the atomic-resolution structure of a riboswitch whose effector is coenzyme B12. To address these research goals, a combination of structural approaches including X-ray crystallography and small angle X-ray scattering, biochemical approaches such as chemical probing and transcriptional assays, and bioinformatics. The results of these proposed studies will serve to broaden our knowledge of RNA-based gene regulation as well as provide an atomic-level understanding of RNAs that are promising targets of antimicrobial agents. PUBLIC HEALTH RELEVANCE: Riboswitches are a form of RNA-based gene regulation that is widely utilized in bacteria, including a number of medically important pathogenic bacteria such as S. aureus, M. tuberculosis, P. aeruginosa and Streptococcus species. Our work seeks to develop an atomic-level understanding of how these RNAs regulate bacterial through their ability to directly bind cellular metabolites. These studies serve to further our understanding into how RNA can be exploited as targets of antibacterial therapeutics via structure-based drug design.